CN111175429A - Method for establishing fingerprint spectrum of bactericidal and antipruritic lotion - Google Patents
Method for establishing fingerprint spectrum of bactericidal and antipruritic lotion Download PDFInfo
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- CN111175429A CN111175429A CN202010015491.2A CN202010015491A CN111175429A CN 111175429 A CN111175429 A CN 111175429A CN 202010015491 A CN202010015491 A CN 202010015491A CN 111175429 A CN111175429 A CN 111175429A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8686—Fingerprinting, e.g. without prior knowledge of the sample components
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
Abstract
The invention provides a method for establishing a fingerprint spectrum of a bactericidal antipruritic lotion, which comprises the following steps: s1, preparation of a mixed reference solution: precisely weighing matrine, astilbin, phellodendrine, berberine hydrochloride, osthole and atractylodin reference substances respectively, and adding methanol to obtain mixed reference substance solution; s2, preparing a sterilization and itching-relieving lotion sample solution; s3, preparing a single reference medicinal material solution; and S3, measuring. The fingerprint maps mark 16 common peaks and determine 5 medicinal materials belonging to the prescription, the established fingerprint maps have high technical content, and the singleness and one-sidedness of the existing quality control method are avoided. Provides effective quality control basis for the production of the bactericidal antipruritic lotion and lays a foundation for the research on the correlation between the drug effect substance basis and the pharmacological action.
Description
Technical Field
The invention relates to the field of medicine detection, in particular to a method for establishing a fingerprint spectrum of a sterilizing and itching-relieving lotion.
Background
The bactericidal itching-relieving lotion is prepared from 6 traditional Chinese medicines of phellodendron, radix sophorae flavescentis, fructus cnidii, rhizoma smilacis glabrae, borneol and rhizoma atractylodis, has the effects of clearing heat, removing toxicity, killing parasites and relieving itching, and is suitable for improving symptoms such as leukorrhagia, pruritus vulvae and the like caused by trichomonas vaginitis, mycotic vaginitis, non-specific vaginitis and pruritus vulvae.
The existing research establishes a C fingerprint spectrum technology of the bactericidal and antipruritic lotion as in HPL fingerprint spectrum research of the bactericidal and antipruritic lotion, but only two characteristic peaks of matrine and sophocarpine are investigated, and characteristic peaks of other medicinal materials such as golden cypress, common cnidium fruit, glabrous greenbrier rhizome and rhizoma atractylodis are not attributed. The components of the bactericidal antipruritic lotion are complex, and only taking the sophora flavescens as a quality control index is obviously incomplete and has certain limitations. Therefore, the development and establishment of a fingerprint detection method which is simple, convenient, feasible, high in accuracy and good in repeatability and can reflect the multiple components contained in the compound preparation as much as possible is very necessary for the quality control and evaluation of the sterilizing and itching-relieving lotion.
Disclosure of Invention
The invention aims to provide a method for establishing a fingerprint of a bactericidal and antipruritic lotion, which aims to solve the problems, 16 common peaks are calibrated, 5 medicinal materials belonging to a prescription are determined, the established characteristic spectrum has high technical content, and the singleness and one-sidedness of the existing quality control method are avoided.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a method for establishing a fingerprint spectrum of a bactericidal antipruritic lotion comprises the following steps:
s1, preparation of a mixed reference solution:
precisely weighing matrine, astilbin, phellodendrine, berberine hydrochloride, osthole and atractylodin reference substances, respectively, and adding methanol to obtain mixed reference substance solution.
S2, preparation of a sterilization and itching-relieving lotion sample solution:
accurately weighing the sterilizing and itching relieving lotion, adding absolute methanol, extracting, filtering, and taking supernatant to obtain a test solution.
S3, preparing a single reference medicinal material solution:
pulverizing cortex Phellodendri, radix Sophorae Flavescentis, fructus Cnidii, rhizoma Smilacis Glabrae and rhizoma Atractylodis into powder, respectively collecting each single medicinal material powder, precisely weighing, adding anhydrous methanol, extracting, filtering, and collecting supernatant to obtain cortex Phellodendri reference medicinal material solution, radix Sophorae Flavescentis reference medicinal material solution, fructus Cnidii reference medicinal material solution, rhizoma Smilacis Glabrae reference medicinal material solution, and rhizoma Atractylodis reference medicinal material solution.
S4, determination:
precisely absorbing and mixing the reference solution and the test solution respectively, injecting into a high performance liquid chromatograph, recording chromatogram, and processing the chromatogram by fingerprint software to obtain the fingerprint of the sterilizing and itching relieving lotion.
Preferably, the detection conditions include: the chromatographic column is a Waters Xbridge C18 chromatographic column; performing gradient elution by taking phosphoric acid aqueous solution with volume fraction of 0.1% as a mobile phase A and acetonitrile as a mobile phase B, wherein the flow rate of the mobile phase is 0.8-1.2 mL/min; the column temperature is 20-35 ℃, the detection wavelength is 285nm, and the sample injection amount is 5-15 mul.
Preferably, the flow rate of the mobile phase is 0.8 mL/min; the column temperature was 25 ℃ and the amount of sample was 10. mu.l.
Preferably, during the gradient elution, the changes of the mobile phase a and the mobile phase B are as follows:
0min, mobile phase A85%, mobile phase B15%;
15min, mobile phase A64% and mobile phase B36%;
22min, mobile phase A60% and mobile phase B40%;
45min, 50% of mobile phase A and 50% of mobile phase B;
50min, mobile phase A35% and mobile phase B65%;
55min, mobile phase A24% and mobile phase B76%;
70min, mobile phase A18% and mobile phase B82%.
Preferably, in step S2, the bactericidal and antipruritic lotion is precisely weighed, absolute methanol is added, ultrasonic extraction is performed for 30min, cooling is performed, vacuum filtration is performed, and 13000r/min supernatant is centrifuged for 10min to obtain a test solution.
Preferably, in step S3, the golden cypress, the radix sophorae flavescentis, the fructus cnidii, the rhizoma smilacis glabrae and the rhizoma atractylodis are crushed into powder, each single medicinal material powder is precisely weighed, absolute methanol is added, ultrasonic extraction is carried out for 30min, cooling is carried out, vacuum filtration is carried out, supernatant fluid is taken and centrifuged for 10min at 13000r/min, and each single control medicinal material solution is respectively obtained.
Preferably, the common peaks of the fingerprint are subjected to drug attribution, and 16 common peaks of the fingerprint are determined, wherein the 1 st, 10 th, 11 th and 15 th peaks belong to a fructus cnidii drug, the 4 th, 5 th and 8 th peaks belong to a rhizoma smilacis glabrae drug, the 7 th, 9 th, 13 th and 14 th peaks belong to a cortex phellodendri drug, the 3 rd, 6 th and 12 th peaks belong to a radix sophorae flavescentis drug, and the 2 nd and 16 th peaks belong to a rhizoma atractylodis drug.
Preferably, the common peaks of the fingerprint are subjected to active ingredient attribution, wherein the peak 3 is matrine, the peak 4 is astilbin, the peak 7 is phellodendrine, the peak 9 is berberine hydrochloride, the peak 10 is osthole, and the peak 16 is atractyloin.
Due to the adoption of the technical scheme, the invention has the following beneficial effects:
1. the fingerprint of the sterilizing and itching-relieving lotion established by the invention marks 16 common peaks, determines 5 medicinal materials belonging to the prescription, has high technical content of the established characteristic spectrum, and avoids the singleness and one-sidedness of the existing quality control method. Provides effective quality control basis for the production of the bactericidal antipruritic lotion and lays a foundation for the research on the correlation between the drug effect substance basis and the pharmacological action.
2. The fingerprint of the sterilizing and itching-relieving lotion established by the invention has 16 common peaks, and identification and attribution analysis are carried out on the common peaks, so that the separation effect of each characteristic peak is good, the quality information of the product can be reflected comprehensively, the comprehensive monitoring of the product quality is facilitated, and the authenticity of the product can be identified. The active ingredients corresponding to 6 peaks in the 16 peaks are confirmed, so that the fingerprint peaks are effectively associated with the treatment efficacy, and the production quality of the sterilizing and itching-relieving lotion can be further controlled.
In 16 common peaks of the fingerprint, peaks 1, 10, 11 and 15 belong to fructus Cnidii, wherein peak 10 is osthole; peak 4, 5 and 8 belong to rhizoma Smilacis Glabrae, wherein Peak 4 is astilbin; no. 7, 9, 13 and 14 peaks belong to cortex Phellodendri medicinal materials, wherein No. 7 peak is phellodendrine, and No. 9 peak is berberine hydrochloride; no. 3, No. 6 and No. 12 peak belong to lightyellow sophora root medicinal materials, wherein the No. 3 peak is matrine; no. 2 and No. 16 peaks belong to rhizoma atractylodis medicinal materials, wherein the No. 16 peak is atractylodin. Therefore, the main components (except that the components of the borneol have no ultraviolet absorption) of the medicinal materials of the sterilizing and itching relieving lotion are integrally represented in the fingerprint spectrum obtained by the technology.
3. The invention relates to the establishment of a fingerprint of a bactericidal and antipruritic lotion, which adopts a high performance liquid chromatogram method to qualitatively and quantitatively detect the bactericidal and antipruritic lotion, determines the optimal chromatographic condition according to the characteristics of active ingredients of the bactericidal and antipruritic lotion, and obtains an accurate fingerprint. Wherein, the mobile phase selects 0.1 percent phosphoric acid water and acetonitrile, and the gradient elution is carried out by continuously changing the proportion of each phase in the mobile phase according to the program, so that the separation degree between chromatographic peaks of each component is good, the interference is small, the peak shape is good, the base line is stable, and the acid solution with lower concentration is beneficial to protecting the chromatographic column.
Drawings
FIG. 1 is a chromatogram comparing different wavelengths in an experimental example of the present invention;
in the figure, 1: 220 nm; 2: 240 nm; 3: 260 nm; 4: 285 nm; 5: 290 nm; 6: 300 nm; 7: 320 nm; 8: 340 nm;
FIG. 2 is a chromatogram of different columns compared in an experimental example of the present invention;
in the figure, 1: waters XBridge C18; 2: cometex C18-AA; 3: ZORBAX Eclipse Plus; 4: zorbax SB; 5: phenomenex luna;
FIG. 3 is a chromatogram comparing different mobile phases for an experimental example of the present invention;
in the figure, 1: pure water-acetonitrile; 2: 0.1% phosphoric acid water-acetonitrile; 3: 0.05% aqueous formic acid-acetonitrile; 4: 0.04% triethylamine buffer-acetonitrile;
FIG. 4 is a chromatogram comparing different column temperatures for experimental examples of the present invention;
in the figure, 1: 35 ℃; 2: 30 ℃; 3: 25 ℃;
FIG. 5 is a chromatogram comparing different flow rates for an experimental example of the present invention;
in the figure, 1: 1.2m L/min; 2: 1.0m L/min; 3: 0.8m L/min;
FIG. 6 is a chromatogram comparing different extraction solvents in the experimental examples of the present invention;
in the figure, 1: 30% methanol; 2: 50% methanol; 3: 80% methanol; 4: anhydrous methanol;
FIG. 7 is a chromatogram comparing different extraction methods according to the experimental example of the present invention;
in the figure, 1: heating and refluxing; 2: carrying out ultrasound;
FIG. 8 is a chromatogram comparing different extraction times in the experimental examples of the present invention;
in the figure, 1: 60 min; 2: 45 min; 3: 30 min; 4: 15 min;
FIG. 9 is a HPLC overlay of samples of bactericidal antipruritic lotion of Experimental example 10 in accordance with the present invention;
FIG. 10 is an HPLC chromatogram of a control mixed solution of an experimental example of the present invention;
in the figure, 1, matrine; 2. astilbin; 3. phellodendrine; 4. berberine hydrochloride; 5. osthole; 6. atractylodin;
FIG. 11 is an HPLC overlay of a control drug according to an experimental example of the present invention;
in the figure, 1, 2, 3, 4, 5, 6 and atractylodes are shown.
FIG. 12 is a fingerprint of the bactericidal antipruritic lotion of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to the following embodiments and the accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Materials:
the apparatus used in the examples of the invention is shown in table 1 below.
TABLE 1 Instrument
The reagent used in the embodiment of the invention is as follows:
matrine, astilbin, phellodendrine, berberine hydrochloride, osthole and atractylodin standard substances are purchased from China food and drug testing research institute. The purity of each standard product is more than 98.0 percent.
Sample preparation: 10 batches of bactericidal and antipruritic lotion (batch numbers 20190301, 20190302, 20190303, 20190304, 20190305, 20190601, 20190602, 20190603, 20190604 and 20190605) and single medicinal materials of golden cypress, lightyellow sophora root, common cnidium fruit, glabrous greenbrier rhizome and swordlike atractylodes rhizome are provided by Guizhou Changsheng pharmaceutical industry Co., Ltd.
Acetonitrile (chromatographically pure), formic acid (chromatographically pure) was purchased from sigma, usa.
Example 1
A method for establishing a fingerprint spectrum of a bactericidal antipruritic lotion comprises the following steps:
s1, preparation of a mixed reference solution:
taking appropriate amount of matrine, astilbin, phellodendrine, berberine hydrochloride, osthole, and atractylodin reference substances, precisely weighing, and adding methanol to obtain mixed reference substance solutions with concentrations of 248.3, 190.7, 154.1, 198.5, 251.4, and 183.6 μ g/mL respectively.
S2, preparation of a sterilization and itching-relieving lotion sample solution:
accurately weighing the sterilizing and itching relieving lotion, adding absolute methanol, carrying out ultrasonic extraction for 30min, cooling, carrying out vacuum filtration, taking supernatant fluid and centrifuging for 10min at 13000r/min to obtain a test solution.
S4, preparing a single reference medicinal material solution:
pulverizing cortex Phellodendri, radix Sophorae Flavescentis, fructus Cnidii, rhizoma Smilacis Glabrae and rhizoma Atractylodis into powder, collecting powder of each single medicinal material about 0.2g, precisely weighing, adding anhydrous methanol, ultrasonic extracting for 30min, cooling, vacuum filtering, collecting supernatant, centrifuging at 13000r/min for 10min, and respectively obtaining test solution of cortex Phellodendri, radix Sophorae Flavescentis, fructus Cnidii, rhizoma Smilacis Glabrae and rhizoma Atractylodis.
S4, measuring
Precisely absorbing and mixing the reference solution and the test solution respectively, injecting into a high performance liquid chromatograph, recording chromatogram, and processing the chromatogram by fingerprint software to obtain the fingerprint of the sterilizing and itching relieving lotion.
The detection conditions include: the chromatographic column is a Waters Xbridge C18 chromatographic column; performing gradient elution by taking phosphoric acid aqueous solution with volume fraction of 0.1% as a mobile phase A and acetonitrile as a mobile phase B, wherein the flow rate of the mobile phase is 0.8 mL/min; the column temperature was 25 ℃, the detection wavelength was 285nm, and the sample size was 10. mu.l.
During the gradient elution process, the changes of the mobile phase A and the mobile phase B are as follows:
0min, mobile phase A85%, mobile phase B15%;
15min, mobile phase A64% and mobile phase B36%;
22min, mobile phase A60% and mobile phase B40%;
45min, mobile phase A50%, mobile phase B50%;
50min, mobile phase A35% and mobile phase B65%;
55min, mobile phase A24% and mobile phase B76%;
70min, mobile phase A18% and mobile phase B82%.
Experimental example:
1. examination of extraction conditions
1.1 investigation of wavelength
Under the same conditions, chromatograms at different wavelengths were compared, and the results are shown in fig. 1.
Wherein, 1: 220 nm; 2: 240 nm; 3: 260 nm; 4: 285 nm; 5: 290 nm; 6: 300 nm; 7: 320 nm; 8: 340 nm.
The results show that: and comprehensively considering the aspects of chromatographic peak number, peak intensity, baseline stability, separation effect and the like, and determining 285nm as the final detection wavelength.
1.2 investigation of the column
Taking the same sterilization antipruritic lotion sample solution, and respectively examining the separation effects of the following 5 different C18 chromatographic columns under the same chromatographic conditions, wherein the separation effects comprise: waters Xbridge C18 (4.6X 250mm,5 μm); cometex C18-AA (250 mm. times.4.6 mm,5 μm); ZORBAX Eclipse Plus C18 (4.6X 250mm,5 μm); zorbax SB-C18 (4.6X 250mm,5 μm); phenomenex luna C18 (4.6X 250mm,5 μm); the results are shown in FIG. 2.
In fig. 2, high performance liquid fingerprint spectra 1 under different chromatographic columns: waters XBridge C18; 2: cometex c 18-AA; 3: ZORBAX Eclipse Plus; 4: zorbax SB; 5: phenomenex luna.
The result shows that the chromatographic profiles with different peak patterns, retention times and separation effects obtained by different chromatographic columns under the same chromatographic conditions are adopted, and the Waters Xbridge C18 chromatographic column can better separate the chemical components of the bactericidal and antipruritic lotion comprehensively considering all aspects, so that the fingerprint chromatogram determination of the bactericidal and antipruritic lotion sample is completed by using the chromatographic column.
1.3 investigation of the mobile phase
The experiment was examined for different mobile phase systems and the results are shown in figure 3.
In the figure 3, high performance liquid phase fingerprint spectrums under different mobile phases are as follows:
1: pure water-acetonitrile; 2: 0.1% phosphoric acid water-acetonitrile; 3: 0.05% aqueous formic acid-acetonitrile; 4: 0.04% Triethylamine buffer-acetonitrile (pH adjusted to 6.5 with phosphoric acid)
The result shows that under the condition of using 0.1 percent of phosphoric acid water-acetonitrile solvent as the mobile phase, the chromatographic peak has better separation degree, so the solvent is selected as the mobile phase.
1.4 examination of column temperature
Examining the effects of different column temperatures, including: the results are shown in FIG. 4 at 25 deg.C, 30 deg.C and 35 deg.C.
High performance liquid chromatograms at different column temperatures in fig. 4, 1: 35 ℃; 2: 30 ℃; 3: at 25 ℃.
The results show that the chromatographic separation effect is not very different, so the room temperature of 25 ℃ with the maximum protection to the chromatographic column is selected as the optimal column temperature.
1.5 investigation of flow Rate
The influence of different flow rates (0.8m L/min, 1.0m L/min and 1.2m L/min) on the separation effect of the chromatographic fingerprint of the bactericidal and antipruritic lotion is examined, and the result is shown in figure 5.
High performance liquid chromatogram at different flow rates in fig. 5 1: 1.2m L/min; 2: 1.0m L/min; 3: 0.8m L/min.
The results showed that the number of peaks was small at a flow rate of 1.2m L/min, and compared with the peaks in the chromatographic spectrum of the sample at flow rates of 0.8m L/min and 1.0m L/min, the number of peaks, the pattern of peaks, the degree of separation, etc. were not significantly different, so that 0.8m L/min, which is a small amount of solvent consumption, was selected as the optimum flow rate.
In conclusion, through a series of condition optimization, the optimal chromatographic conditions of the sterilizing and itching-relieving lotion are finally determined as follows: a Waters Xbridge C18 (4.6X 250mm,5 μm) column was used as a column and the mobile phase was 0.1% phosphoric acid in water (A) -acetonitrile (B) with the following gradient:
0min, mobile phase A85%, mobile phase B15%;
15min, mobile phase A64% and mobile phase B36%;
22min, mobile phase A60% and mobile phase B40%;
45min, 50% of mobile phase A and 50% of mobile phase B;
50min, mobile phase A35% and mobile phase B65%;
55min, mobile phase A24% and mobile phase B76%;
70min, mobile phase A18% and mobile phase B82%.
The volume flow is 0.8m L/min; the detection wavelength is 285 nm; the column temperature is 25 ℃; the amount of the sample was 10. mu.L.
2. Extraction condition optimization
2.1 selection of extraction solvent
Accurately weighing 4 parts of bactericidal and antipruritic lotion sample, each 2ml, pouring into a conical flask, respectively adding 50ml of anhydrous methanol, 80% methanol, 50% methanol and 30% methanol as solvents, performing ultrasonic extraction, filtering and collecting filtrate, centrifuging the sample solution at 13000r/min for 10min, extracting 10 mu L of the sample solution, injecting into a high performance liquid chromatograph, and collecting a chromatogram (figure 6).
High performance liquid chromatograms of different extraction solvents in fig. 6 1: 30% methanol; 2: 50% methanol; 3: 80% methanol; 4: anhydrous methanol.
The result shows that the chromatogram obtained by using the anhydrous methanol as the extraction solvent has the most chromatographic peaks, better resolution and larger peak area value of each peak, which indicates that the extraction of the anhydrous methanol is more complete, and the anhydrous methanol is determined to be the extraction solvent.
2.2 selection of extraction mode
Accurately weighing 2 parts of bactericidal and antipruritic lotion sample, wherein each 2ml of bactericidal and antipruritic lotion sample is obtained by respectively adopting two different extraction methods of ultrasonic and reflux, filtering and collecting filtrate, centrifuging the sample at 13000r/min for 10min, extracting 10 mu L of the filtrate, injecting the sample into a high performance liquid chromatograph, and collecting a chromatogram (figure 7).
High performance liquid chromatograms of different extraction methods in fig. 7 1: heating and refluxing; 2: ultrasound
The results show that the two extraction methods have similar effects, and because the operation of ultrasonic extraction is more convenient and easy and the reproducibility is good, the sterilization and itching-relieving lotion is extracted by the ultrasonic method.
2.3 extraction time selection
Accurately weighing 4 parts of bactericidal and antipruritic lotion sample, each 2ml, placing the sample in a conical flask, respectively performing ultrasonic extraction for 15min, 30min, 45min and 60min by using anhydrous methanol as a solvent, filtering and collecting filtrate, centrifuging the sample solution for 10min under the condition of 13000r/min, extracting 10 mu L of the sample solution, injecting the sample solution into a high performance liquid chromatograph, and collecting a chromatogram (figure 8).
FIG. 8 is a high performance liquid chromatogram of different extraction times
1：60min；2：45min；3：30min；4：15min。
The results show that the chromatogram obtained at different extraction times are similar in effect, and in order to save manpower and material resources and ensure that the sample is completely extracted, ultrasonic extraction is finally selected for 30 min.
In summary, the final extraction process was determined to be: adding anhydrous methanol, ultrasonic extracting for 30min, cooling, vacuum filtering, collecting supernatant, centrifuging at 13000r/min for 10min to obtain sample solution.
3. Methodology investigation
3.1 precision test
Taking an S1 sample, preparing a sample solution according to the extraction method of the embodiment 1, collecting sample information according to the chromatographic method of the embodiment 1, continuously injecting samples of 6 needles, and introducing HPLC (high performance liquid chromatography) spectrum data of the samples injected for 6 times into a Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition) for similarity calculation, wherein the similarity of the results is more than 0.99. The 9 th peak with good separation degree and moderate peak area is taken as a reference peak, the RSD of the relative retention time of 16 common peaks is calculated to be less than 0.05 percent, the RSD of the relative peak area is calculated to be less than 2 percent, and the result is shown in table 1. The precision of the instrument used in the experiment is better, the analysis result is stable and reliable, and the requirement of the fingerprint spectrum is met.
3.2 stability test
Taking an S1 sample, preparing a sample solution according to the extraction method in the embodiment 1, collecting sample information according to the chromatographic method in the embodiment 1, injecting the sample information into a liquid chromatograph for 0h, 2h, 4h, 8h, 12h and 24h respectively, introducing the HPLC fingerprint data of the samples in 6 time periods into a similarity evaluation system for calculation, and obtaining the result that the similarity of the samples in the 6 time periods is more than 0.99. Relative retention time RSD of 16 common peaks was calculated to be < 0.20% and relative peak area RSD < 3% using peak No. 9 as the reference peak, and the results are shown in table 2. Indicating that the sample was stable over 24 h.
3.3 repeatability test
Precisely weighing the sample No. S1, preparing 6 parts of test solution in parallel, and introducing the HPLC fingerprint data of the 6 parts of sample into a similarity evaluation system for calculation, wherein the similarity of the 6 batches of samples is more than 0.96. The relative retention time RSD of 16 common peaks is calculated to be less than 0.3 percent by taking the peak No. 9 as a reference peak, and the RSD of the relative peak area is calculated to be less than 3.09 percent, which indicates that the method has good repeatability. The results are shown in Table 2.
TABLE 2 analytical results
4. Establishment of fingerprint
Respectively and precisely absorbing 10 batches of the sterilization and itching-relieving lotion sample solution, the reference substance mixed solution and each single reference medicinal material solution by 10 mu L, injecting the sample into a high performance liquid chromatograph according to the chromatographic method in the embodiment 1, and recording the chromatogram. The results are shown in FIGS. 9, 10 and 11.
Wherein, FIG. 9 is an HPLC overlay chart of 10 batches of bactericidal antipruritic lotion samples.
FIG. 10 shows HPLC chromatogram 1 of control mixed solution and matrine; 2. astilbin; 3. phellodendrine; 4. Berberine hydrochloride; 5. osthole; 6. atractylodin is used.
FIG. 11 is an HPLC overlay of control drug. 1. Characteristic fingerprint 2, glabrous greenbrier rhizome 3, common cnidium fruit 4, amur corktree bark 5, lightyellow sophora root 6 and swordlike atractylodes rhizome.
5. Establishment of common fingerprint and similarity evaluation
And (3) importing 10 batches of sample HPLC chromatogram data into a similarity evaluation system, automatically matching by using a median method, performing multipoint correction to form 16 common peak spectrums with stable retention time and obvious peak shapes as sample common characteristic HPLC fingerprints (see figure 12), and calculating similarity results shown in table 3 to indicate that the HPLC spectrums in all batches have good similarity.
TABLE 310 calculation of sample similarity for bactericidal and antipruritic lotion batches
| S1 | S2 | S3 | S4 | S5 | S6 | S7 | S8 | | S10 | |
| S1 | ||||||||||
| 1 | 0.992 | 0.992 | 0.985 | 0.985 | 0.99 | 0.99 | 0.992 | 0.985 | 0.992 | |
| S2 | 0.992 | 1 | 0.991 | 0.983 | 0.982 | 0.992 | 0.992 | 0.999 | 0.982 | 0.988 |
| S3 | 0.992 | 0.993 | 1 | 0.983 | 0.982 | 0.992 | 0.992 | 0.991 | 0.982 | 0.978 |
| S4 | 0.985 | 0.983 | 0.983 | 1 | 0.989 | 0.976 | 0.976 | 0.983 | 0.976 | 0.983 |
| S5 | 0.985 | 0.982 | 0.982 | 0.987 | 1 | 0.976 | 0.975 | 0.982 | 0.921 | 0.982 |
| S6 | 0.99 | 0.992 | 0.992 | 0.976 | 0.976 | 1 | 0.976 | 0.992 | 0.976 | 0.992 |
| S7 | 0.99 | 0.992 | 0.992 | 0.976 | 0.975 | 0.988 | 1 | 0.992 | 0.975 | 0.992 |
| S8 | 0.992 | 0.987 | 0.987 | 0.983 | 0.982 | 0.992 | 0.992 | 1 | 0.982 | 0.934 |
| S9 | 0.985 | 0.982 | 0.982 | 0.988 | 0.985 | 0.976 | 0.975 | 0.982 | 1 | 0.982 |
| S10 | 0.992 | 0.978 | 0.987 | 0.983 | 0.982 | 0.992 | 0.992 | 0.954 | 0.982 | 1 |
| R | 0.995 | 0.997 | 0.997 | 0.992 | 0.992 | 0.993 | 0.993 | 0.997 | 0.992 | 0.997 |
6, identification and attribution of common peaks of fingerprint spectra:
according to retention time information of chromatographic peaks, performing medicinal material attribution on common peaks of a fingerprint, and determining 16 common peaks of the fingerprint, wherein the No. 1, No. 10, No. 11 and No. 15 peaks belong to a fructus cnidii medicinal material, and the No. 10 peak is osthole; peak 4, 5 and 8 belong to rhizoma Smilacis Glabrae, wherein Peak 4 is astilbin; no. 7, 9, 13 and 14 peaks belong to cortex Phellodendri medicinal materials, wherein No. 7 peak is phellodendrine, and No. 9 peak is berberine hydrochloride; no. 3, No. 6 and No. 12 peak belong to lightyellow sophora root medicinal materials, wherein the No. 3 peak is matrine; no. 2 and No. 16 peaks belong to rhizoma atractylodis medicinal materials, wherein the No. 16 peak is atractylodin. Therefore, the main components (except that the components of the borneol have no ultraviolet absorption) of the medicinal materials of the sterilizing and itching relieving lotion are integrally represented in the fingerprint spectrum obtained by the technology.
The above description is intended to describe in detail the preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the claims of the present invention, and all equivalent changes and modifications made within the technical spirit of the present invention should fall within the scope of the claims of the present invention.
Claims (8)
1. The method for establishing the fingerprint spectrum of the bactericidal antipruritic lotion is characterized by comprising the following steps of:
s1, preparation of a mixed reference solution:
precisely weighing matrine, astilbin, phellodendrine, berberine hydrochloride, osthole and atractylodin reference substances respectively, and adding methanol to obtain mixed reference substance solution;
s2, preparation of a sterilization and itching-relieving lotion sample solution:
accurately weighing the sterilizing and itching relieving lotion, adding anhydrous methanol, extracting, filtering, and taking supernatant to obtain a test solution;
s3, preparing a single reference medicinal material solution:
pulverizing cortex Phellodendri, radix Sophorae Flavescentis, fructus Cnidii, rhizoma Smilacis Glabrae and rhizoma Atractylodis into powder, respectively taking each single medicinal material powder, precisely weighing, adding anhydrous methanol, extracting, filtering, and taking supernatant to obtain cortex Phellodendri reference medicinal material solution, radix Sophorae Flavescentis reference medicinal material solution, fructus Cnidii reference medicinal material solution, rhizoma Smilacis Glabrae reference medicinal material solution, and rhizoma Atractylodis reference medicinal material solution;
s4, determination:
precisely absorbing the mixed reference solution, the test solution and each single reference medicinal material solution, respectively, injecting into a high performance liquid chromatograph, recording chromatogram, and processing the chromatogram with fingerprint software to obtain the fingerprint of the lotion for sterilizing and relieving itching.
2. The method for establishing the fingerprint of the bactericidal antipruritic lotion according to claim 1, wherein the detection conditions comprise: the chromatographic column is a Waters Xbridge C18 chromatographic column; performing gradient elution by taking phosphoric acid aqueous solution with volume fraction of 0.1% as a mobile phase A and acetonitrile as a mobile phase B, wherein the flow rate of the mobile phase is 0.8-1.2 mL/min; the column temperature is 20-35 ℃, the detection wavelength is 285nm, and the sample injection amount is 5-15 mul.
3. The method for establishing the fingerprint of the bactericidal antipruritic lotion according to claim 2, wherein the flow rate of the mobile phase is 0.8 mL/min; the column temperature was 25 ℃ and the amount of sample was 10. mu.l.
4. The method for establishing the fingerprint of the bactericidal antipruritic lotion according to claim 1, wherein the changes of the mobile phase A and the mobile phase B in the gradient elution process are as follows:
0min, mobile phase A85%, mobile phase B15%;
15min, mobile phase A64% and mobile phase B36%;
22min, mobile phase A60% and mobile phase B40%;
45min, 50% of mobile phase A and 50% of mobile phase B;
50min, mobile phase A35% and mobile phase B65%;
55min, mobile phase A24% and mobile phase B76%;
70min, mobile phase A18% and mobile phase B82%.
5. The method for establishing the fingerprint of the bactericidal and antipruritic lotion according to claim 1, wherein in step S2, the bactericidal and antipruritic lotion is precisely weighed, absolute methanol is added, ultrasonic extraction is performed for 30min, cooling is performed, vacuum filtration is performed, and 13000r/min of supernatant is taken and centrifuged for 10min to obtain a test solution.
6. The method for establishing the fingerprint of the bactericidal and antipruritic lotion according to claim 1, wherein in step S3, the golden cypress, the radix sophorae flavescentis, the fructus cnidii, the rhizoma smilacis glabrae and the rhizoma atractylodis are pulverized into powder, each single medicinal material powder is respectively taken and precisely weighed, absolute methanol is added, ultrasonic extraction is carried out for 30min, cooling is carried out, vacuum filtration is carried out, supernatant fluid is taken and centrifuged for 10min at 13000r/min, and each single control medicinal material solution is respectively obtained.
7. The method for establishing the fingerprint of the bactericidal antipruritic lotion according to any one of claims 1 to 6, wherein the common peaks of the fingerprint are subjected to drug attribution, and the 16 common peaks of the fingerprint are determined, wherein the 1 st, 10 th, 11 th and 15 th peaks belong to the medicinal material of cnidium fruit, the 4 th, 5 th and 8 th peaks belong to the medicinal material of glabrous greenbrier rhizome, the 7 th, 9 th, 13 th and 14 th peaks belong to the medicinal material of phellodendron bark, the 3 rd, 6 th and 12 th peaks belong to the medicinal material of sophora flavescens, and the 2 nd and 16 th peaks belong to the medicinal material of atractylodes rhizome.
8. The method for establishing fingerprint of bactericidal antipruritic lotion according to any one of claims 1 to 6, wherein the common peaks of the fingerprint are assigned as active ingredients, wherein peak 3 is matrine, peak 4 is astilbin, peak 7 is phellodendrine, peak 9 is berberine hydrochloride, peak 10 is osthole, and peak 16 is atractyloin.
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